The repair of lesions in DNA is accomplished by a variety of mechanisms in mammalian cells. Perhaps the two most well-studied of these pathways are nucleotide excision repair and base excision repair. In contrast, recombinational repair pathways that repair double-strand breaks and interstrand crosslinks have not, as yet, been as thoroughly investigated. For interstrand crosslink repair in particular, a major impediment to progress has been the lack of an in vitro cell-free assay which would allow a biochemical analysis of this pathway. In this proposal, we show that such an assay has been developed with mammalian cell-free extracts. Results from the assay correlate very strongly with the known sensitivity of various cell lines to crosslinking agents. This assay will be utilized to determine the factors required in the assay, to investigate the mechanisms of recombinational repair of interstrand crosslinks, and to begin the biochemical fractionation of factors that are involved in this pathway. Finally, in addition to these studies, two novel human genes, that are homologues of the yeast RAD51/RAD55/RAD57 family, have been obtained from a private sequence database. These new RecA-like genes will be characterized with particular emphasis on their possible role in crosslink repair.